I’d like to stain for apoptosis markers cleaved capase3 or cleaved PARP on denaturating western blots. There are multiple antibodies available in the market that claim to recognize the cleaved fragment but not the full length protein. But how is this possible? The cleaved fragment is part of the full protein, thus the epitope present in the cleaved fragment is also present in the full-length protein… and I guess conformation should not be contributing under denaturating conditions.
I can´t prove it but conformation must be the key. Denaturation doesn´t mean that you get a string of amino acids like pearls on a chain. The protein structure will change during the heating and it is possible that due to the cleavage other conformations will appear. Antibodies against this conformation will be specific. But you are right in challenging the claims of antibody producers!
Antibodies against cleaved Caspase 3 do work pretty well. On a western blot you can see the size difference so it is not even a big deal if it recognizes the uncleaved form. I believe the epitopes are very close to the cleavage site and are masked in the full length protein but exposed in the cleaved form.
Some proteins expose the antigenic domains upon cleavage and may this is happening in this case.
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